1. Field of the Invention
The present invention, in the areas of microbiology and immunology, relates to a novel lectin with binding specificity toward sialic acid, to methods of its production and isolation, and to its uses in diagnosis and treatment of diseases. The lectin may be isolated from Tritrichomonas species.
2. Description of the Background Art
The discovery of microbial lectins has opened new approaches for studies of host-parasite interactions. These carbohydrate-specific molecules appear to play an important role in cell recognition and cell adhesion (Liener et al., The Lectins. Properties, Functions and Applications in Biology and Medicine, Academic Press, New York (1986)). Many species of bacteria as well as some viruses produce lectins (Mirelman (ed.), Microbial Lectins and Agglutinins: Properties and Biological Activity, Wiley, New York (1986)). Very few lectins from parasitic protozoa have been characterized extensively. These include Giardia lamblia (Farthing et al., Infect. Immun. 51:661-667 (1986): Lev et al., Science 232:71-73 (1986); Lev et al., In: Mirelman (ed.) Microbial Lectins and Agglutinins: Properties and Biological Activity, Wiley, New York (1986)) and Entamoeba histolytica (Kobiler et al., Infect. Immun. 29:211-225 (1980); Mirelman et al., In: Mirelman (ed.), Microbial Lectins and Agglutinins: Properties and Biological Activity, Wiley, New York (1986); Rosales-Encina et al., J. Infect. Dis. 156:790-797 (1987)).
Sialic acid is a carbohydrate produced by both prokaryotic and eukaryotic cells. Due to the ubiquity of sialic acid on the surface of cells of prokaryotic as well as eukaryotic origin, and the presence of sialic acid on a large proportion of glycoproteins in the serum of mammals and in other biological fluids, a stable lectin with high affinity for sialic acid would be of great utility for purposes of diagnosis and therapy of a large variety of diseases.
Many types of carcinomas produce specialized glycoproteins, the majority of which are sialoglycoproteins (Sharma et al., Indian J. Pathol. Microbiol. 30:317 (1987)). Among the large variety of human carcinomas producing high molecular weight sialoglycoproteins, such as mucins, human metastatic colon carcinoma cells were found to produce 4 tumor-associated sialoglycoproteins (Irimura et al., J. Cell. Biochem. 37:1 (1988)). Various tumors have higher concentrations of sialic acid on their surface than do normal cells, and some tumors secrete polysialic acid (Roth et al., Amer. J. Pathol. 133:227-240 (1988); Echenique et al., Urology 32:397-400 (1988); Ravindranaths et al., J. Biol. Chem. 263:2079-2086 (1988)).
Surface sialic acid expression in the form of sialogangliosides is also characteristic of tumors, such as melanoma (Cheresh et al., Proc. Natl. Acad. Sci. USA 82:5155-5159 (1985)); see for review: Dyatlovitskaya et al., Biochim. Biophys. Acta 907:125-143 (1987)).
Direct and indirect approaches exist for localization of sialic acid in tissues. In indirect procedures, tissues are processed in parallel with and without neuraminidase digestion to remove sialic acid. Tissues are then stained based on surface negative charge, with reagents such as, for example, colloidal iron hydroxide (Gasic et al., J. Cell. Biol. 19:223 (1963)) and cationized ferritin (Danon et al., J. Ultrastruct. Res. 38:500 (1972). Comparison of tissue bearing, and denuded of, sialic acid is then made, in order to determine the localization of sialoglycoproteins. These procedures are of limited specificity.
A number of lectins have been shown to have some specificity for terminally sialylated glycoproteins. Wheat germ agglutinin used in conjunction with its succinylated derivatives has been used to localize sialic acid on colon tissue in a relatively nonspecific manner (Moore et al., Amer. J. Pathol. 131:477 (1988)).
Sialic acid-specific lectins have been isolated from the hemolymph of several horseshoe crabs, Limulus polyphemus (Marchalonis et al., J. Mol. Biol. 32:453 (1968)) and Carcinoscorpus rotunda (Biochim. Biophys. Acta 623:89 (1980)) as well as from lobster and sponge. Of these, only the Limulus lectin is commercially available. This lectin is of very high molecular weight, has a tendency to dissociate (Marchalonis et al., J. Mol. Biol. 32:453 (1968)), and is quite expensive ($100/mg; Sigma Chemicals, Catalog).
In addition, a large number of viruses have hemagglutinin activity which is based on apparent binding to sialic acid (Mirelman (ed.), Microbial Lectins and Agglutinins: Properties and Biological Activity, Wiley, New York (1986)). None of these is commercially available.
Miller (U.S. Pat. Nos. 4,457,865 and 4,520,111) disclosed a sialic acid specific lectin from the slug Limax flavus. This is the only marketed lectin reagent which has been tested for its utility in detecting sialic acid on cells and tissues. For example, Roth et al. (J. Histochem. Cytochem. 32:1167-1176 (1984)) used the Limax flavus lectin to investigate the distribution of sialic acid in rat pancreas, liver, and colonic mucosa, using fetuin-gold to visualize the tissue-bound lectin. However, the slug lectin is difficult to isolate, expensive, and more importantly, its availability is limited by the organism from which it is derived, since the supply of slugs is limited and depends on unpredictable factors such as weather.
A plant lectin isolated from the bark of the elderberry (Sambucus nigra L.), termed SNA (EY Scientific, San Mateo, Calif.), binds to sialic acid linked to either galactose or N-acetyl galactosamine, in an .alpha.2-6 linkage (Shibuya et al., J. Biol. Chem. 202:1596-1601 (1987)). This lectin has been used to affinity purify glycoproteins, oligosaccharides, and glycopeptides possessing the appropriate disaccharide sequence (Shibuya et al., Arch. Biochem. Biophys. 254:1-8 (1987)), although it was found that lectin binding was lost when branching side chains existed at the O-3 position of the penultimate N-acetyl galactosamine residue. This property may limit the usefulness of this lectin.
In summary, very few useful reagents are available in large quantities and at reasonable cost which can serve as sufficiently specific agents to identify, characterize, or even treat tumors on the basis of cell surface sialic acid expression. Therefore, the need for a sialic acid-specific lectin of microbial origin which can be produced economically in large quantities, which is stable, specific, and of high affinity, is well-recognized in the art.
Furthermore, isolation and characterization of sialic acid binding lectin(s) from protozoa would be of benefit in our understanding of the pathobiology of parasite-host interactions. Such a lectin in appropriate pharmacologic form could be employed to modify adherence of the protozoa to host tissues, thereby disrupting infection by Tritrichomonas species. Production of monoclonal antibodies against these trichomonad lectins would provide both diagnostic and therapeutic tools for parasitology.